Semimicrochemical Assay for Diethylstilbestrol C. W. SONDERN AND CLARENCE BURSON, George A. Breon & Co., Kansas City, Mo.
S
INCE the discovery of diethylstilbestrol (alpha, alpha’diethyl-4,4’-stilbenediol) by Dodds et al. (2) in England,
the use of this potent estrogen has spread throughout the world. The bioassay of diethylstilbestrol b y the well-known Allen and Doisy method is both time-consuming and uneconomical; therefore a chemical procedure is needed which will serve as a n analytical tool. [Since this paper was prepared a colorimetric method for the assay of diethylstilbestrol has been TABLE I. BROMINATION OF KNOWN AMOUNTS OF DIETHYLSTILBESTROL
Weight of Sample
1 1 2 2 5 5 10 10 10 40 40 40 40
(Dried a t 100’ C . ) Diethylstilbestrol Found
0.960 0.982 2.05 1.94 4.98
4.9I
5.00 9.92 9.81 10.18 39.81 40.06 40.06 40.37
Recovery
96.0 98.2 102.5 97.0 9.. 9 .. 6 .
98.2 100.0 99.2 9S.l 101.2 99.55 100.15 100.15 100.93
described b y Dechne, (f).] Sprung (4) and Francis and Hill (3) describe a bromometric titration method for phenols, using standard bromide-bromate solution which may be applied to this drug. Bromination of known quantities of diethylstilbestrol (Table I) has established that the method is suitable for determining diethylstilbestrol in the amounts normally found in pharmaceutical preparations (1 to 40 mg.)
Method
OIL SOLUTIONS OF DIETHYLSTILBESTROL. Accurately measure a sample containing 5 mg. of diethylstilbestrol into a separatory funnel, add 30 cc. of light petroleum ether, and extract 4 times with 2.5 per cent sodium hydroxide solution, using two 10cc. and two 5-cc. portions. Pass the extractions through a small funnel containing a plug of cotton into a 125-cc. glass-stoppered iodination flask. Add 5 cc. of 0.1 N bromide-bromate solution and proceed as directed for the tablets, beginning with “Quickly add 5 cc. of hydrochloric acid”. GLYCEROGELATINSUPPOSITORIES OF DIETHYLSTILBESTROL. Dissolve in hot water a sufficient quantity of the suppositories to yield 5 mg. of diethylstilbestrol, cool, and acidulate with dilute sulfuric acid. Extract 4 times with ether, using 20-cc., 15-cc., 15-cc., and 10-cc. portions, and pass through a funnel containing a plug of cotton into a suitable vessel. Evaporate the extract nearly to dryness and pass steam over the residue for 1 hour to volatilize and remove any henolic preservative present in the gelatin. Evaporate most or the water and dissolve the residue in 2.5 per cent sodium hydroxide. Transfer to an iodination flask, add 5 cc. of 0.1 N bromide-bromate solution, and proceed as directed for the tablets, beginning with “Quickly add 5 cc. of hydrochloric acid”.
FACTOR IN BROMINATION OF TABLE 11. TEMPERATURE-TIME DIETHYLSTILBESTROL c.
Time
Bromine Used
Min.
Atom8
5-10 5-10 5-10 5-10 15 15 20 20 25 25 30 30 30 60
60 180 380 960 15 30 15 30 15 30 15 30 360 60
9.9 10.0 10.2 11.6 9.5 11.0 10.0 11.2 11.0 12.0 12.0 12.0 12.0 12.0
Temperature
From the brominations shown in Table I1 i t is evident that diethylstilbestrol in a cold solution first removes ten atoms of bromine from solution. The removal of two atoms more takes place slowly in the cold, rapidly at higher temperatures. The removal of ten atoms can be made consistent by controlling the time and temperature, but since twelve atoms will eventually be removed if the reaction goes to completion i t seems more logical to brominate for 30 minutes a t above 25’ C.
A N A L Y s I s OF TABLETS. Weigh not less than 20 tablets; reduce them to a fine powder, and place an accurately weighed aliquot equivalent to about 10 mg. of diethylstilbestrol in a micro-Soxhlet extractor. Extract with ether for 1 hour, transfer the extract to a 125-cc. glass-stoppered iodination flask, and evaporate to dryness on a water bath. Dissolve the residue in 5 cc. of warm aqueous 2.5 er cent sodium hydroxide solution, cool, and add 10 cc. of staniard U. S. P. XI bromide-bromate solution (0.1 N bromine solution) by means of a pipet. Discussion Quickly add 5 CC. of hydrochloric acid, insert the stopper, and place 5 cc. of 10 per cent potassium iodide solution around the Under the conditions described in the analytical procedure, stopper. Set aside in a dark place for 30 minutes at 25’ to 30” C. twelve atoms of bromine enter into the reaction, accompanied Remove the stopper just sufficiently to introduce the potassium by the release of 5 moles of hydrogen bromide for each mole iodide solution and shake the flask thoroughly, taking care that no bromine vapor escapes. Carefully rinse the stopper and the neck of the flask with a little distilled OH OH OH OH water and then titrate the liberated B r A B r Br/\Br iodine with 0.05. N sodium thiosulfate solution, using starch indicator at the end of the titration. Each cubic centimeter of bromidebromate solution is equivalent to 2.233 mg. of diethylstilbestrol. The presence of material capable of bromination will seriously affect Brq at the accuracy of the assay-for example, 60 mg. of stearic acid U.S. P. Br 300 c.at , B r k BrBrfiBr HBr may contain sufficient oleic acid as Dried impurity to introduce an error of 8 50” c. C-c per cent in a determination of 10 mg. of diethylstilbestrol. Consequently, /I I BrAH &2HS HC CzH, a blank determination on the filler and lubricant material in tablets I I CHa CH3 should be made. 358
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+
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ANALYTICAL EDITION
April 15, 1942
of diethylstilbestrol. Presumably the bromination product contains seven atoms of bromine. Studies made with 4,4'dihydroxystilbene indicate that four Of these are located in the 3,5- and 3',5'-positions. It would be expected that simultaneously two atoms of bromine would add at the double bond. The splitting out of one of these bromine atoms as hydrogen bromide would provide a vinylene linkage into enter. The diagram tmo more bromine atoms appears to explain the data in the most satisfactory manner.
359
Literature Cited (1) Dechne, C. B., J. Am. Pharm. Assoc., 30, 208-9 (1941).
(2) Dod&, E. C., Goldberg, L., Lawson, W., and Robinson, R., Nature, 141, 247 (1938). (3) Francis, A. W., and Hill, A. J., J. Am. Chem. Soc., 46, 2498 (1924); IND.ENG.CHEM.,ANAL.ED., 13, 357 (1941). Ibid., 13, 35 (1941). (4) Sprung, Jf. M., PRESENTED before the Division of Medicinal Chemistry a t the 102nd hleeting of the h V E R 1 C A N CHEDIIC4L SOCIETY, ~ t ~ a n t City, i c N. J.
Separation of Copper, Lead, and Zinc with Salicvlaldoxime J
L. P. BIEFELD AND W. B. LIGETT Purdue University, Lafayette, Ind.
T
HE separation of copper from other metals which form
complexes with salicylaldoxime is possible because copper salicylaldoximate may be precipitated in weakly acidic solutions, whereas the salicylaldoximates of most other metals are precipitated only from neutral or slightly basic solutions (1-6, IO). Lead has been separated from silver, zinc, and cadmium b y precipitation in strongly ammoniacal solution (7). T h e effect of hydrogen-ion concentration upon the precipitation of zinc with salicylaldoxime has been reported briefly ( 5 ) . The purpose of the present investigation was to make a more detailed study of the effect of p H on the precipitation of zinc salicylaldoximate, and to determine the best conditions for a separation of copper, lead, and zinc based upon p H control and ammonia-complex formation.
Precipitation of Zinc Salicylaldoximate The fact that zinc forms a n insoluble complex with salicvlaldoxime was first reported b y Ephraim studied the properties of the precipitate and concluded t h a t i t was not suitable for the quantitative determination of zinc because the p H range for complete precipitation was too narrow, the precipitate was appreciably soluble in various neutral salt solutions, and the compound slomly decomposed above 80" C. The compound precipitated b y Pearson contained 19.45 per cent zinc by analysis and evidently mas Zn(C7H602S)2, which contains 19.36 per cent zinc. Flagg and Furman (5) found that if zinc salicylaldoximate, together with the solution from which i t has been freshly precipitated, is warmed to 90" C. for 10 minutes, a compound corresponding t o Zn(C;H602N) results. This compound, containing 32.61 per cent zinc, can be dried at 110" C. without decomposition. Since the compound obtained by Flagg and Furman is obviously more suitable for quantitative purposes, the work described below is based upon precipitation of the compound Zn(CiH50zS).
(e).
REAGENTSAND APPAR.4TvS. The salicylaldoxime used was obtained from the Eastman Kodak Company. A 1 per cent solution was prepared bv dissolving 1 gram of the reagent in 5 ml. of alcohol and slomly pouring the alcohol
solution into 95 ml. of water warmed to 80" C. The reagent solution was cooled and filtered before use. The zinc solution was prepared from reagent grade zinc nitrate. It was standardized by precipitation and weighing as Zn?\'H4P04,and by precipitation as ZnSH4P04and ignition to ZnPZOr. , A glass electrode pH meter as described by Mellon (8) was used. I t was calibrated with Clark and Lubs buffer solutions. The reagents used in standardization of the zinc solution, those used in preparation of the buffers, and the ammonia used for adjustment of pH were all of reagent grade. Calibrated weights and volumetric ware were used. Redistilled water was employed for the preparation of all solutions, for dilutions, and for washing the precipitates. PRECIPITATION AND pH MEASUREMENT. I n each case, 25 ml. of a 1 per cent salicylaldoxime solution were added dropwise to 25 ml. of zinc solution containing 0.0999 gram of zinc and ammonia in an amount estimated to give the desired pH. Water was added to make 100 ml. The mixture was mechanically stirred during addition of the reagent. After all the reagent had been added, solution and precipitate were heated to about 90" C. for 10 minutes, cooled to room temperature, filtered with suction on KO.4 Jena glass crucibles, washed with water, and dried a t 110" C. for 1 hour. Ferric chloride solution was used to test for complete washing of the precipitate, thorough washing
PH FIGURE1. PRECIPITATION OF COPPER, LEAD,ASD Zrsc
